Synergistic Adhesive Interactions and Signaling Mechanisms Operating between Platelet Glycoprotein Ib/IX and Integrin αIIbβ3: STUDIES IN HUMAN PLATELETS AND TRANSFECTED CHINESE HAMSTER OVARY CELLS

Cindy L Yap,Sascha C Hughan,Susan L Cranmer,Warwick S Nesbitt,Michael M Rooney,Simon Giuliano,Suhasini Kulkarni,Sacha M Dopheide,Yuping Yuan,Hatem H Salem,Shaun P Jackson

doi:10.1074/jbc.m005590200

Abstract

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin alpha(IIb)beta(3). These include the following: 1) examining the sufficiency of GPIb/IX and integrin alpha(IIb)beta(3) to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin alpha(IIb)beta(3) activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin alpha(IIb)beta(3), we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin alpha(IIb)beta(3) mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin alpha(IIb)beta(3). Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin alpha(IIb)beta(3) activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150-1800 s(-)(1)). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin alpha(IIb)beta(3) activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin alpha(IIb)beta(3) activation.

Highlights

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin ␣IIb␤3
Studies on human platelets demonstrated that von Willebrand factor (vWf) binding to GPIb/IX is able to induce integrin ␣IIb␤3 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150 –1800 s؊1)
This multivalent adhesive interaction is unique in that it can tether platelets at high shear stresses [6], a key requirement for the ability of platelets to secure hemostasis throughout the arterial circulation. vWf binding to GPIb/IX induces platelet activation, converting the major platelet integrin, ␣IIb␤3, from a low affinity to a high affinity receptor capable of engaging the C1 domain of vWf (8 –10)

Summary

STUDIES IN HUMAN PLATELETS AND TRANSFECTED CHINESE HAMSTER OVARY CELLS*

A major unresolved issue is the mechanism by which the vWf-GPIb interaction induces activation of integrin ␣IIb␤3 under physiological flow conditions It is unclear whether GPIb/IX induces integrin ␣IIb␤3 activation directly, through the generation of intracellular second messengers, or involves an indirect pathway dependent on the release of ADP and/or the generation of thromboxane A2 (TXA2). Evidence favoring the latter mechanism has been suggested from studies of shear-induced platelet aggregation using a cone-and-plate viscometer [13, 14], in which the exposure of platelets in suspension to pathological levels of shear induces platelet activation in an ADP-dependent manner According to this model (Fig. 1), shear-induced binding of soluble vWf to GPIb induces transmembrane calcium influx through an unidentified surface channel functionally linked to the GPIb/IX complex. We have demonstrated that intracellular calcium mobilization and activation of protein kinase C (PKC) are two key signaling events linking the vWf-GPIb interaction to integrin ␣IIb␤3 activation over the full range of shear forces experienced by platelets in vivo

EXPERIMENTAL PROCEDURES

RESULTS

Previous studies have demonstrated that transfection of

DISCUSSION